1. The Field of the Invention
The invention relates to apparatus and methods for fractionating microstructures such as free cells, viruses, macromolecules, or minute particles. More particularly, the present invention relates to apparatus and methods for sorting such microstructures in suspension in a fluid medium while simultaneously viewing individual of those microstructures during the process.
2. Background Art
The sizing, separation, and study of microstructures such as free cells, viruses, macromolecules, and minute particles are important tools in molecular biology. For example, this fractionation process when applied to DNA molecules is useful in the study of genes and ultimately in planning and the implementation of genetic engineering processes. The fractionation of larger microstructures, such as mammalian cells, promises to afford cell biologists new insights into the functioning of these basic building blocks of living creatures.
A. Macromolecule Fractionation
While many types of macromolecules may be fractionated by the apparatus of the present invention, the fractionation of a DNA molecule will be discussed below in detail as one example.
The DNA molecules in a single cell of a complex organism contain all of the information required to replicate that cell and the organism of which it is a part. A DNA molecule is a double helical chain of four different subunits that occur in a genetically coded succession along the chain. The four subunits are the nitrogenous bases, adenine, cytosine, guanine, and thymine. The size of such a molecule is measured by the number of such bases it contains. Natural DNA molecules range in size from a few kilobasepairs in length to hundreds of megabasepairs in length. The size of a DNA molecule is roughly proportional to the number of genes the molecule contains.
The size of a DNA molecule can also be expressed by its molecular weight, its length, or the number of basepairs it includes. If the number of basepairs is known, that number can be converted into both the length and the molecular weight of the DNA molecule. One method for estimating the size of small DNA molecules is the process of gel electrophoresis.
In gel electrophoresis an agarose gel is spread in a thin layer and allowed to harden into a firm composition. The composition comprises a fine network of fibers retaining therewithin a liquid medium, such as water. The fibers of the agarose gel cross and interact with each other to form a lattice of pores through which molecules smaller than the pores may migrate in the liquid retained in the composition. The size of the pores in the lattice is determined generally by the concentration of the gel used.
Slots are cast in one end of the gel after the gel is hardened, and DNA molecules are placed into the slots. A weak electric field of typically 1-10 volts per centimeter is then generated in the gel by placing the positive pole of an electric power source in one end of the gel and the negative pole of the power source in the opposite end. In DNA electrophoresis, the negative pole of the power source is placed in the gel at the end of the composition in which the slots containing the DNA are located. The DNA molecules, being negatively charged, are induced by the electric field to migrate through the gel to the positive pole of the power source at the other end of the composition. This occurs at speeds of typically only a few centimeters per hour.
The electrophoretic mobility of the molecules can be quantified. The electrophoretic mobility of a molecule is the ratio of the velocity of the molecule to the intensity of the applied electric field. In a free solution, the mobility of a DNA molecule is independent of the length of the molecule or of the size of the applied electric field. In a hindered environment, however, aside from the structure of the hindered environment, the mobility of a molecule becomes a function of the length of the molecule and the intensity of the electric field.
The gels used in gel electrophoresis is just such a hindered environment. Molecules are hindered in their migration through the liquid medium in the gel by the lattice structure formed of the fibers in the gel. The molecules nevertheless when induced by the electric field, move through the gel by migrating through the pores of the lattice structure. Smaller molecules are able to pass through the pores more easily and thus more quickly than are larger molecules. Thus, smaller molecules advance a greater distance through the gel composition in a given amount of time than do larger molecules. The smaller molecules thereby become separated from the larger molecules in the process. In this manner DNA fractionation occurs.
While gel electrophoresis is a well known and often used process for DNA fractionation, electrophoretic mobility is not well understood in gel lattice structures. Thus, the process has several inherent limitations. For example, the pore size in the lattice of gels cannot be accurately measured or depicted. Therefore, the lengths of the molecules migrating through the lattice cannot be accurately measured. It has also been found that DNA molecules larger than 20 megabasepairs in length cannot be accurately fractionated in gels. Apparently, the pore size in the lattice of such materials cannot be increased to permit the fractionation of larger molecules, much less even larger particles, viruses, or free cells.
Further, the lattice structure formed when a gel hardens is not predictable. It is not possible to predict the configuration into which the lattice structure will form or how the pores therein will be positioned, sized, or shaped. The resulting lattice structure is different each time the process is carried out. Therefore, controls and the critical scientific criteria of repeatability cannot be established.
Gel electrophoresis experiments cannot be exactly duplicated in order to predictably repeat previous data. Even if the exact lattice structures formed in one experiment were determinable, the structure could still not be reproduced. Each experiment is different, and the scientific method is seriously slowed.
Also, the lattice structure of a gel is limited to whatever the gel will naturally produce. The general size of the pores can be dictated to a degree by varying the concentration of the gel, but the positioning of the pores and the overall lattice structure cannot be determined or designed. Distinctive lattice structures tailored to specific purposes cannot be created in a gel.
Further, because the lattice structure arrived at depends upon the conditions under which hardening of the gel occurs, the lattice structure even in a single composition need not be uniform throughout.
Another shortcoming of gel electrophoresis is caused by the fact that a gel can only be disposed in a layer that is relatively thick compared to the pores in its lattice structure, or correspondingly to the size of the DNA molecules to be fractionated. Thus, the DNA molecules pass through a gel in several superimposed and intertwined layers. Individual DNA molecules cannot be observed separately from the entire group. Even the most thinly spread gel is too thick to allow an individual DNA molecule moving through the gel to be spatially tracked or isolated from the group of DNA molecules.
Once a gel has been used in one experiment, the gel is contaminated and cannot be used again. The gel interacts with the materials actually used in each experiment, and cleaning of the gel for later reuse is not possible. A gel layer must therefore be disposed after only one use. This also frustrates the scientific objective of repeatability.
Finally, simple gel electrophoresis cannot be used to fractionate DNA molecules larger than approximately 20 kilobases in length. To overcome this fact, it is known to pulse the applied electric field to attempt to fractionate longer DNA molecules. This technique, however, results in extremely low mobility and requires days of running time to achieve significant fractionation. Also, the numerical predictions of the theories developed to explain the results of this technique depend critically on the poorly known pore size and distribution in the lattice of the gel.
B. Cell Fractionation
The flexibility of cells is a structural variable of some interest to cell biologists. The flexibility of cells and the effects of various environments on cell flexibility is important to the study of the aging process in cells. However, cell fractionation based upon cell flexibility is not easily accomplished in the prior art.
For example, various cells have round or oval shapes with various diameters. The shapes are often determined by an underlying cytoskeleton.
When the cells are circulating in the human body, the cells must, on several occasions, pass through variously sized openings and passageways. This requires substantial flexibility of the cell. The inability to pass through these openings can be caused by the aging of a cell, reactions to specific chemical environments, and other metabolic changes. When referring to red blood cells, poor red blood cell flexibility results in serious consequences for the larger organism. With respect to cells such as cancer cells, poor flexibility may result in growth and spread of tumors.
Cancer cells are generally thought to settle in the human body in blood vessels larger than the cells themselves and stick to those vessels through a special adhesion molecule. As the cancer cells stick to the vessels, new tumors begin to grow. New information, however, has indicated that the cancer cells move too quickly to become adhered to the vessels in this fashion. It is now thought that cells may start new tumors when they become stuck in vessels too narrow for the cancer cells to pass through. The flexibility of the cancer cells is important in determining the deleterious effect of the cell.
Three physical limitations impinge on the flexibility of many cells. First, many cells must maintain both a constant volume V and a constant surface area A as it deforms. Second, the cell membrane, while very flexible, cannot increase in area. It will tear, if forced to do so. Third, as a cell ages it loses membrane and the surface-to-volume ratio decreases.
For example, a biconcave red blood cell has a maximum diameter of about 8 microns, a surface area of about 140 microns square, and a volume of about 95 microns cube in the normal state. It can be shown that for mature red blood cells for openings smaller in diameter than approximately 3 microns, the constraints of constant volume V and surface area A cannot be met. The passage of a red blood cell through a passageway of that size, thus, cannot occur without membrane rupture. Since the smallest capillary openings are but approximately 3.5 microns, red blood cells passing through the capillary bed are uncomfortably close to being ruptured. Accordingly, small changes in the physical variables that control deformability can lead to microangiopathy and severe organism distress.
There exist several techniques for measuring cell flexibility and deformability. These range from the elegant and pioneering micropipette aspiration techniques, to the nucleopore filtration and laminar stress elongation techniques. The latter are termed ektacytometry. All are very useful and have provided an excellent initial database for studying red blood cell deformation, but each has certain weaknesses.
The micropipette aspiration technique can only study one cell at a time. The nucleopore filtration technique does not allow observation of cells during the actual passage thereof through openings. Ektacytometry does not deform cells in narrow passages.